1. Technical Field
The field of this invention is the use of chimeric surface membrane proteins for signal transduction.
2. Background
Regulation of cell activities is frequently achieved by the binding of the ligand to a surface membrane receptor. The formation of the complex with the extracellular portion of the receptor results in a change in conformation with the cytoplasmic portion of the receptor undergoing a change which results in a signal being transduced in the cell. In some instances, the change in the cytoplasmic portion results in binding to other proteins, where the other proteins are activated and may carry out various functions. In some situations, the cytoplasmic portion is autophosphorylated or phosphorylated, resulting in a change in its activity. These events are frequently coupled with secondary messengers, such as calcium, cyclic adenosine monophosphate, inositol phosphate, diacylglycerol, and the like. The binding of the ligand results in a particular signal being induced.
There are a number of instances, where one might wish to have a signal induced by virtue of employing a different ligand. For example, one might wish to activate particular T-cells, where the T-cells will then be effective as cytotoxic agents, or activating agents by secretion of interleukins, colony stimulating factors or other cytokines, which results in the stimulation of another cell. The ability of the T-cell receptor to recognize antigen is restricted by the nature of Major Histocompatibility Complex (MHC) antigens on the surface of the host cell. Thus, the use of a chimeric T-cell receptor in which a non-MHC restricted ligand binding domain is linked directly to the signal transducing domain of the T-cell receptor would permit the use of the resulting engineered effector T-cell in any individual, regardless of their MHC genetic background. In this manner, one may change the ligand which initiates the desired response, where for some reason, the natural agent may not be as useful.
There is, therefore, interest in finding ways to modulate cellular responses in providing for the use of ligands other than the normal ligand to transduce a desired signal.
Relevant Literature
The T-cell antigen receptor (TCR) has a non-covalent association between a heterodimer, the antigen/MHC binding subunit Ti variable component and the five invariant chains: zeta (xcex6), eta (xcex7) and the three CD3 chains: gamma (xcex3), delta (xcex4) and epsilon (xcex5) (Weiss and Imboden (1987) Adv. Immunol., 41:1-38; Cleavers et al. (1988) Ann. Rev. Immunol., 6:629-662; Frank et al. (1990) Sem. Immunol., 2:89-97). In contrast to the Ti alpha/beta heterodimer which is solely responsible for antigen binding, the physically associated CD3-zeta/eta complex does not bind ligand, but is thought to undergo structural alterations as a consequence of Ti-antigen interaction which results in activation of intracellular signal transduction mechanisms.
A description of the zeta chain may be found in Ashwell and Klausner (1990) Ann. Rev. Immunol., 8:139-167. The nature of the zeta chain in the TCR complex is described by Baniyash et al. (1988) J. Biol. Chem., 263:9874-9878 and Orloff et al. (1989) ibid., 264:14812-14817. The heterodimeric zeta and eta protein is described by Jin et al. (1990) Proc. Natl. Acad. Sci. USA, 87:3319-3323. Discussion of the homo- and heterodimers may be found in Mercep et al. (1988) Science, 242:571-574; and Mercep et al. (1989) ibid., 246:1162-1165. See also Sussman et al. (1988) Cell, 52:85-95. For studies of the role of the zeta protein, see Weissman et al. (1989) EMBO, J., 8:3651-3656; Frank et al. (1990) Science, 249:174-177; and Lanier et al. (1989) Nature, 342:803-805.
For discussion of the gamma subunit of the Fcxcex5 R1 receptor, expressed on mast cells and basophils and its homology to the zeta chain, see Bevan and Cunha-Melo (1988) Prog. Allergy, 42:123-184; Kinet (1989) Cell, 57:351-354; Benhamou et al., Proc. Natl. Acad. Sci. USA, 87:5327-5330; and Orloff et al. (1990) Nature, 347:189-191.
The zeta(xcex6) chain is structurally unrelated to the three CD3 chains, and exists primarily as a disulfide-linked homodimer, or as a heterodimer with an alternatively spliced product of the same gene, eta (xcex7). The zeta chain is also expressed on natural killer cells as part of the Fcxcex3RIII receptor. The gamma chain of the Fcxcex5 receptor is closely related to zeta, and is associated with the Fcxcex5RI receptor of mast cells and basophils and the C16 receptor expressed by macrophages and natural killer cells. The role in signal transduction played by the cytoplasmic domains of the zeta and eta chains, and the gamma subunit of the FcRI receptor has been described by Irving and Weiss (1991) Cell 64:891-901; Romeo and Seed, (1991) Cell 64:1037-1046 and Letourneur and Klausner (1991) Proc. Natl. Acad. Sci. USA 88:8905-8909. More recent studies have identified an 18 amino-acid motif in the zeta cytoplasmic domain that, upon addition to the cytoplasmic domain of unrelated transmembrane proteins, endows them with the capacity to initiate signal transduction (Romeo et al. (1992) Cell 68:889-897). These data suggest a T cell activation mechanism in which this region of zeta interacts with one or more intracellular proteins.
The three CD3 chains, gamma (xcex3), delta (xcex4) and epsilon (xcex5), are structurally related polypeptides and were originally implicated in signal transduction of T cells by studies in which anti-CD3 monoclonal antibodies were shown to mimic the function of antigen in activating T cells (Goldsmith and Weiss (1987) Proc. Natl. Acad. Sci. USA 84:6879-6883), and from experiments employing somatic cell mutants bearing defects in TCR-mediated signal transduction function (Sussman et al. (1988) Cell 52:85-95). Sequences similar to the active motif found in zeta are also present in the cytoplasmic domains of the CD3 chains gamma and delta. Chimeric receptors in which the cytoplasmic domain of an unrelated receptor has been replaced by that of CD3 epsilon have been shown to be proficient in signal transduction (Letourneur and Klausner (1992) Science 255:79-82), and a 22 amino acid sequence in the cytoplasmic tail of CD3 epsilon was identified as the sequence responsible. Although the cytoplasmic domains of both zeta and CD3 epsilon have been shown to be sufficient for signal transduction, quantitatively distinct patterns of tyrosine phosphorylation were observed with these two chains, suggesting that they may be involved in similar but distinct biochemical pathways in the cell.
The phosphatidylinositol-specific phospholipase C initiated activation by the T-cell receptor (xe2x80x9cTCRxe2x80x9d) is described by Weiss et al. (1984) Proc. Natl. Acad, Sci. USA, 81:416-4173; and Imboden and Stobo (1985) J. Exp. Med., 161:446-456. TCR also activates a tyrosine kinase (Samelson et al. (1986) Cell, 46:1083-1090; Patel et al. (1987) J. Biol. Chem., 262:5831-5838; Chsi et al. (1989) J. Biol. Chem., 264:10836-10842, where the zeta chain is one of the substrates of the kinase pathway (Baniyash et al. (1988) J. Biol. Chem., 263:18225-18230; Samelson et al. (1986), supra). Fyn, a member of the src family of tyrosine kinases, is reported to coprecipitate with the CD3 complex, making it an excellent candidate for a TCR-activated kinase (Samelson et al. (1990) Proc. Natl. Acad. Sci. USA, 87:4358-4362). In addition, a tyrosine kinase unrelated to fyn has been shown to interact with the cytoplasmic domain of zeta (Chan et al., (1991) Proc. Natl. Acad. Sci. USA, 88:9166-9170).
Letourner and Klausner (1991) Proc. Natl. Acad. Sci. USA 88: 8905-8909 describe activation of T cells using a chimeric receptor consisting of the extracellular domains of the xcex1 chain of the human interleukin 2 receptor (Tac) and the cytoplasmic domain of either xcex6 or xcex3. Gross et al., (1989) Proc. Natl. Acad. Sci. USA 86: 10024-10028 describe activation of T cells using chimeric receptors in which the MHC-restricted antigen-binding domains of the T cell receptor xcex1 and xcex2 chains were replaced by the antigen-binding domain of an antibody. Romeo and Seed (1991) Cell 64: 1037-1046 describe activation of T-cells via chimeric receptors in which the extracellular portion of CD4 is fused to the transmembrane and intracellular portions of xcex3, xcex6, and xcex7 subunits. Letourner and Klausner (1992) describe activation of T cells by a chimeric receptor consisting of the extracellular domain of the IL-2 receptor and the cytoplasmic tail of CD3 epsilon (Science 255:79-82).
Based on the structural similarities between the immunoglobulin (Ig) chains of antibodies and the alpha (xcex1) and beta (xcex2) T cell receptor chains (Ti), chimeric Ig-Ti molecules in which the V domains of the Ig heavy (VH) and light (VL) chains are combined with the C regions of Ti xcex1 and Ti xcex2 chains have been described (Gross et al. (1989) Proc. Natl. Acad. Sci. USA, 86:1002-10028). The role of the Ti chains is to bind antigen presented in the context of MHC. The Ti heterodimer does not possess innate signalling capacity, but transmits the antigen-binding event to the CD3/zeta chains present in the TCR complex. Expression of a functional antigen-binding domain required co-introduction of both VH-Ti and VL-Ti chimeric molecules. These chimeras have been demonstrated to act as functional receptors by their ability to activate T cell effector function in response to cross-linking by the appropriate hapten or anti-idiotypic antibody (Becker et al. (1989) Cell, 58:911 and Gross et al. (1989) Proc. Natl. Acad. Sci. USA 86:10024). However, like the native Ti chains, the VH-Ti and VL-Ti chains do not possess innate signalling capacity, but act via the CD3/zeta complex.
The triggering of signal transduction leading to cytotoxic function by different cells of the immune system can be initiated by chimeric receptors with antibody type specificity. These chimeric receptors by-pass the requirement for matching at the MHC locus between target cell (i.e. virally infected, tumor cell, etc.) and effector cell (i.e., T cell, granulocyte, mast cell, etc.). Intracellular signal transduction or cellular activation is achieved by employing chimeric proteins having a cytoplasmic region associated with transduction of a signal and activation of a secondary messenger system, frequently involving a kinase, and a non-MHC restricted extracellular region capable of binding to a specific ligand and transmitting to the cytoplasmic region the formation of a binding complex. Particularly, cytoplasmic sequences of the zeta, eta, delta, gamma and epsilon chains of TCR and the gamma chain of Fcxcex5R1, or a tyrosine kinase are employed joined to other than the natural extracellular region by a transmembrane domain, and the cytoplasmic region is not naturally joined to an extracellular ligand-binding domain. In this manner, cells capable of expressing the chimeric protein can be activated by contact with the ligand, as contrasted with the normal mode of activation for the cytoplasmic portion.